In the process of inflammation an initial wave of inflammatory cells, comprised primarily of polymorphonuclear leukocytes (PMN) is soon followed by a second wave of cells, which are predominantly monocytes (Hurley et al.; Wilkinson et al.). Although it is widely held that monocytes arrive at an area of inflammation as a result of chemotaxis (Hayashi et al.), the specific mediator(s) responsible for the recruitment of monocytes has remained unresolved.
Monocytes are derived from pro-monocytes, found in bone marrow. The pro-monocytes differentiate into monocytes which are released into the blood. The monocytes circulate in the blood until they are attracted to the site of injury by the inflammation process. Once monocytes enter into tissue they mature into macrophages, also referred to as mononuclear phagocytes. Macrophages are able to engulf and destroy foreign antigens; accordingly, macrophages play an important role in the body's immunological defense system. The term "monocyte" as used herein refers collectively to both circulating monocytes and to macrophages present in tissue.
The preferential migration of monocytes during the latter phase of inflammation indicates the requirement for highly cell-specific chemoattractant, which has little or no effect on the migration of PMNs. Experiments indicate that a granule-associated cationic protein (mol. wt. 37,000 daltons) from human PMN acts as a monocyte-specific chemoattractant. This protein has been previously referred to in the liter attire as CAP37, cationic antimicrobial protein of mol. wt. 37,000 daltons. CAP37 protein has been previously shown to (i) bind bacterial lipopolysaccharides with a high degree of specificity and affinity, and (ii) possess antimicrobial activity against a number of Gram negative bacteria, such as Salmonella typhimurium and Escherichia coli (Shafer et al., 1986). Thus, CAP37 may play three important functional roles in host defense.
While use of purified CAP37 has far reaching and important functions involving the cellular progression of inflammation, antimicrobial and antineoplastic defenses of the host, the activity of CAP37 as isolated from human PMNs may be limited because of (1) the very small quantities that can be purified and (2) the potential hazards of using human blood products. Use of recombinant CAP37 may overcome some of these problems, but CAP37 is still a large molecule. However, synthetic peptide fragments derived from CAP37 that possess chemotactic, antimicrobial or LPS binding activity and are considerably smaller (e.g., about 25 amino acids in length; approx. 2500-3000 daltons) would overcome these problems. That is, these fragments would be conveniently sized, capable of being produced in unlimited quantities and possess a non-infectious nature.
The instant specification discloses a conventional method for purifying CAP37 to homogeneity and a method for making CAP37 using recombinant DNA techniques. The characterization of the cDNA encoding human CAP37 protein is disclosed. The instant specification has also advanced the art into unchartered areas by disclosing proteins and peptides related and/or derived from CAP37 that have monocyte chemotactic activity, antibiotic activity or lipopolysaccharide (LPS) binding activity. These proteins and peptides can either be synthesized or produced using recombinant DNA techniques. Finally, the instant specification discloses methods for treating diseases and wounds using CAP37 and its related proteins and peptides as well as antibodies to CAP37 and proteins and peptides derived from CAP37 and methods of using these antibodies.